ZM 306416 (CB676475)

VEGFR1 (IC50 = 0.33 μM); Src (IC50 = 0.33 μM); Abl (IC50 = 1.3 μM)

ZM-306416 selective anti-proliferative effect that spares the wild type EGFR cell lines A549 and H2030 (IC50>10 μM) but selectively inhibits the growth of the EGFR-addicted NSCLC cell lines H3255 and HCC4011 (IC50=0.09±0.007 μM and 0.072±0.001 μM, respectively). ZM-306416 is also observed to have a less potent IC50 value of 1.3±0.2 μM toward the ABL kinase, which inhibits the ABL in vitro kinase activity[2].

An assay for luminescence ADP production kinase was utilized to evaluate the activity of confirmed positives in a panel of kinases, which included EGFR, VEGFR1, SRC, and ABL kinase, as previously mentioned. In dose response studies with a 384-well format, the potency of each compound was measured using 12 doubling dilutions in duplicate, with the compound concentrations at 10 μM and 1 μM serving as the upper limit. The PP-384-M Personal Pipettor was used for all reagent transfers. A volume of 1 μL was added to the wells of white 384 well microtiter plates (Corning #3570, Corning, NY) containing the tested compounds or controls. The controls were 30 μM staurosporine in 1% DMSO (v/v) and 1% DMSO (v/v) (high control and low control, respectively). The assay buffer consisted of 25 mM Hepes/NaOH, pH 7.5, 10 mM MgCl2, 2 mM TCEP, 20 mM β-Glycerol Phosphate, and 100 μM Na3VO4. To achieve a final concentration of 50 nM enzyme, 4 μL of kinase dilution in assay buffer was added to each well for each kinase on the panel. It is noteworthy that the concentration of active enzyme in the preparation is unknown due to the unknown specific activity of the panel's kinases. Kinase and the compound were pre-incubated for ten minutes at room temperature following the addition of the enzyme. Afterward, 5 μL of a mixture comprising ATP and Poly(Glu, Tyr) substrates in solution within the assay buffer was introduced into each well, ultimately achieving a 200 μM final concentration. ADP-Glo Reagent (10 μL) was added to each well following a 45-minute reaction at room temperature. 20 microliters of Kinase Detection Reagent were added after 40 minutes of incubation, and another 60 minutes of incubation were required. Using LEADseekerTM, the output of the luminescence signal was measured. The average data from duplicates are represented by the dose response curves, which were plotted using SigmaPlot. The error bars stand for the regression's standard error. Compound IC50 in the assay conditions was calculated with a low limit of 10 nM.

Confirmed hits' anti-proliferative effect was evaluated using a panel of established cell lines, which included A549-EGFRB, A549, and H2030 cell lines harboring wild type EGFR and H3255 and HCC4011 cell lines harboring the activating L858R EGFR mutation. As previously mentioned, A549-EGFRB cells were cultured. As previously mentioned, H2030, H3255, and HCC4011 cells were cultured. F12K medium (Invitrogen Cat.# 21127-022) supplemented with 10% FBS (PAA Cat.# A15-201) was used to cultivate A549 cells. Employing 12 doubling dilutions in duplicate, with compound concentrations of 10 μM and 1 μM as the upper limit, and the Alamar Blue viability assay as previously described, the anti-proliferative effect of each compound was evaluated in dose response studies in 384-well format. A 1% DMSO (v/v) high control and a 1 μM “killer mix” in 1% DMSO (v/v) low control were the contents of the control wells. It took 120 hours for the compound to fully incubate with the cells. The standard error of the regression is represented by the error bars in dose response curves plotted with SigmaPlot 9.0 (Systat Software, San Jose, CA). The mean data from duplicates is shown.

[1]. Synthesis of a novel biotin-tagged photoaffinity probe for VEGF receptor tyrosine kinases. Bioorg Med Chem Lett. 2006 Jan 1;16(1):129-33.

[2]. A high-content biosensor-based screen identifies cell-permeable activators and inhibitors of EGFR function: implications in drug discovery. J Biomol Screen. 2012 Aug;17(7):885-99.

C16H13CLFN3O2

333.74

333.07

C, 57.58; H, 3.93; Cl, 10.62; F, 5.69; N, 12.59; O, 9.59

690206-97-4

196603-47-1 (HCl);690206-97-4;

Solid powder

COC1=C(C=C2C(=C1)C(=NC=N2)NC3=C(C=C(C=C3)Cl)F)OC

YHUIUSRCUKUUQA-UHFFFAOYSA-N

InChI=1S/C16H13ClFN3O2/c1-22-14-6-10-13(7-15(14)23-2)19-8-20-16(10)21-12-4-3-9(17)5-11(12)18/h3-8H,1-2H3,(H,19,20,21)

N-(4-chloro-2-fluorophenyl)-6,7-dimethoxyquinazolin-4-amine

ZM 306416; ZM-306416; CB 676475; CB676475; CB-676475; ZM306416

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.49 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (7.49 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)